Mitochondria are the main sources of energy in the cell. They contain their own DNA (mtDNA), whose genes encode components of the respiratory chain/oxidative phosphorylation system. They are essential for the normal functioning of all cells in the body, and are absolutely critical for the function of those tissues that are highly dependent on aerobic metabolism, especially muscle and brain. Since 1988, both mtDNA point mutations and mtDNA rearrangements (i.e. large-scale deletions [delta-mtDNAs] and duplications [dup-mtDNAs]) have been associated with a heterogeneous group of sporadic, mendelian-, and maternally- inherited mitochondrial encephalomyopathies. This Continuing Competitive Application proposes to follow up on our currently- funded efforts to study mtDNA rearrangements from both a basic and applied point of view. We have two specific aims. We propose to continue our studies of the relationship between wild-type and rearranged mtDNA species, in order to understand fundamental mechanisms of mtDNA rearrangement formation and maintenance, especially as they relate to the pathogenesis of mitochondrial disease. Using cytoplasmic hybrids (cybrids) of human mtDNA-less cells (" rho Angstrom units cells") repopulated with either delta-mtDNAs or dup-mtDNAs, we have made the surprising discovery that cells do not regulate mtDNA copy number on a per-molecule basis, but rather maintain a constant mass of mtDNA. We will now follow up on this result. First, we will study the rate of replication of these unusual mtDNA species. Second, we will also try to determine if dup-mtDNAs can give rise to delta-mtDNAs in these cells; a positive result would provide a mechanistic explanation for the "triplasmy" (wt, delta-, and dup- mtDNAs) found in many patients. Third, we will study the two pairs of replication origins in dup-mtDNAs, to see whether the mode of replication of this molecule can shed light on the copy number issues described above. Finally, we will search for potential factors associated with the control of mtDNA copy number.